Note: Descriptions are shown in the official language in which they were submitted.
~3058~7
1 BACKGROUND OF THE INVENTION
Spergualin is an antibiotic found by Umezawa
tone of the present inventors) et. al. (USP 4,416,~99).
Since its discovery, many Spergualin-related compounds
have been synthesized by Umezawa et. al. (USP 4,529,549,
USP 4,556,735, etc.).
The compounds of the present invention have an
immuno-modulating action with low toxicity and are
expected as a medicine.
Conventional immuno-modulating agent generally
have a strong side effect. Therefore, a compound having
an excellent immuno-modulating action with reduced side
effect and toxicity has been desired.
SUMMARY OF THE INVENTION
An object of the present invention is to provide
an immuno-modulating agent (an immunosuppressive agent
or i~nuno-stimulating agent~ with reduced toxicity.
Immunosuppressive agents are believed to be effective,
for example, for the suppression of immunity required at
the time of organ transplantation or as a thereapeutic
agent for autoimmune diseases. Meanwhile, immuno-
stimulating agents are believed to be useful for the cure
of diseases caused by weakened immunity.
I\cc~o~ illg l~c~ i,hc~ pres~lt in~ntioll th~L-e is
~rov:id~(l a process ~or produciny a novel
Spergu~lin-related compound ~ep~sented b~ the
yeneral fornlul~
1~ R
2 ~C-NH-.Y-(C~)3-CONHCllCONll-(CH~)4-N-(cll2)3 N 2
[Il
(wher~in X is -~CI12)3 5- ~r ~ I ~ is -H or
-CHzoHt Rl is ~ CH-CO- or ~CH3)2cHCH2-CH CO-7
~2 is
) a group repre~ented by the ~ormula
NH
Xl 3~3(CH2)n-CH.~C~7
(w~erein n is 0 or 1 Xl 18 -H cr -OHI ~nd X;~ 18 -H
or -CH20H),
~2) ~ gr~up r~presented by the formula
l4
3 ( CHz ) m ~H CO
(wherein m i6 ~n lnteger oF O to 4~ X3 ~3 ~H,
-COOH, -Ol~, -N~2 or -CONH2~ X4 is -H or -NH2~ an~
~t least on~ of X3 and X4 iB -~H~),
(3) a group represen~ed by the formula
H~
1 ~
~3058~7
~tc~3)2
~wher~lt~ y ls 1 or 2; A is ~ CH-CO- o~
I ~ co_; ~nd, when y i~ Z, two As for~n a pep~:ide
linkage), or
~4) a group represented by the formula
CH -CH -CH-CO-
NH7~ OH
or a pha~a~loglcally acceptable salt thereoi', the
process comprising remo~ing protecting ~roups ~rom
a protecting groups-containing co~pound repre~en~ed
by the general formula [II~
H2N~ ¦3 14
~(cH2j3-coNl`lcl~coNH-(cH2)~-~-(cH2)3-NH-~5
[ ~ I ]
~wherein X has the same definition a~ g~ven
pxeviou~ly; R3 is -H or -CH2-0-Y ~wherein Y ls -H
or a protecting group $or -OH)1 R4 ig -H,
phenylglycyl group wherein the am~no group i~
p~otec~ed, or a leucyl ~roup wherein the amino
group ~s protected~ and R5 i 6 B group oorresFonding
~ to ~2 ill whioh the amino grou~ is protectedl ~nd,
when R4 is a phenylglyoyl or leucyl group wh~.rein
th~ amino group . is protec:ted, R5 is a phenyl or
leucyl gxoup wherein the amino gro~lp ig prote~ctedl
to obtain ~aid Sper~ual~n-related compound.
- lb -
13~5827
'l'he present invention also ~rovides
a Spergualin-~elated ~onlpound repre!lented
by the general formula lII
R R
2 ~C-NH-X-~CH2)3-CONttlllCONH-(CH~)4-1-(CH2)3 NH-R2
[I]
(~hereln X ~ ~CH2)3_$ or ~ ~ R 19 -H o~
-CH20H: Rl ls -H, ~CH-CO- or ~CH3~2CHCH2-CH-C~-
~and R2 13 3ame a~ defined in clalm 1, or
pharmacologl~ally acceptable ~ thereaf.
~ ' .
~ ~ - lc -
'
,
:~30.~27
1 DETAILED DESCRIPTION OF THE INVENTION
As a result of extensive study, the present
inventors found that novel Spergualin-related compounds
represented by the general formula [I~
H2N \ R IRl
~c~NH-x-~cH2)3-coNHcHcoNH-(cH2)4-N-(cH2)3-NH-R2 [I]
(wherein X is -(CH2)3 5- or ~ ; R is -H or -CH2OH;
NH2 NH2
Rl is -H, ~ - CH-CO- or (CH3)2CHCH2-CH-Co-; R2 is a
residue obtained by removing, from an amino acid or
peptide, the hydroxyl group of the carboxyl group and,
when Rl is a group other than -H, R2 is same as Rl) or
pharmacologically acceptable salts thereof have an
excellent activity, low toxicity and hign safety. Based
on this finding, the present invention has been
completed.
As R2 of the general formula [I], there can be
mentioned a hydrogen atom and residues obtained by remov-
ing, from the following amino acids or peptides, the
hydroxyl groups of the carboxyl groups. It is to be
noted that the stereochemical configuration of these
amino acid residues is L, D or DL except glycine, ~-
alanine and y-aminobutyric acid.
-- 2 --
~;~(13~2
1 (1~ Amino acids
Alanine, arginine, ornithine, aspartic aci~,
asparagine, cysteine, cystine, glutamic acid,
glutamine, pyroglutamic acid, glycine, histidine,
lysine, proline, hydroxyproline, isoleucine,
leucine, methionine, phenylalanine, phenyl-
substituted phenylalanine, serine, threonine,
tryptophane, homoserine, tyrosine, valine,
phenylglycine, p-hydroxyphenylglycine,
4-hydroxymethyl-3-hydroxyphenylglycine, ~-alanine,
y-aminobutyric acid, 3-amino-2-hydroxy-4-phenylbutyric
acid, etc.
~2) Peptides
As peptides, di- or tripeptides wherein two to
lS three same or different amino acids mentioned above are
condensed are preferred. There are illustrated the
following examples.
Alanylalanine, leucylleucine, valylvaline,
phenylalanylphenylalanine, tyrosyltyrosine,
phenylglycylphenylglycine, glycylglycine,
isoleucylisoleucine, leucylphenylalanine,
phenylalanylleucine, leucylphenylglycine, phenyl-
glycylleucine, glycylglycylglycine, phenylglycyl-
phenylglycylphenylglycine, phenylalanylphenyl-
alanylphenylalanine, leucylleucylleucine, etc.
Preferable amino acids or peptides are, for
example, phenylglycine, phenylalanine, leucine, aspar~ic
Z7
1 acid, tryptophane and alanine, as well as peptides formed
by the condensation of two to three of said amino acids.
Phenylglycine, phenylalanine, leucylleucine, etc. are
more preferable.
Typical examples of R2 are shown below.
(1) A group represented by the formula
Xl~ NH2
(CH2) n-lH-CO-
(wherein n is 0 or 1; Xl is -H or -OH; and X2 is -H or
--CH2H ) -
(2) A group represented by the formula
x4
X3-(CH2)m-CH-Co-
(wherein m is an integer of 0 to 4; X3 is -H, -COOH,
-OH, -NH2 or -CONH2; X4 is -H or -NH2; and at least
one of X3 and X4 is -NH2).
(3) A group represented by the formula
H=(A)y~
CH2CH(CH3)2
(wherein y is an integer of 1 or 2; A is -NH-CH-CO~
or ~ CQ ; and, when y is 2, two As form a peptide
linkage).
-- 4
~3~ 7
1 (4) A group represented by the formula
CH2 - CH - CH - CO-
Preferable of the compounds represented by
the general formula [I] or their pharmacologically
acceptable salts are, for example, such compounds as in
the general formula [I], X is ~ , R is -CH20H, Rl
NH2
is -H or (L or D) - ~ CH-CO-, (CH3)2CHCH2fH-CO-,
NH2
NH2 NIH2
and R2 is (L or D)- ~ CH-CO-, (L)- ~ -CH2CH-CO-,
NIH2 INH2 NIH2
(L)-HO ~ CH2CH-CO-~ (L) CH3CH-CO-, (L~-(CH3)2CHCH2CH-C9-,
or (L,L~-(CH3)2CHCH CHCONHCH-CO- , or pharmacologically
21
NH2 CH2CH(CH3)2
acceptable salts thereof.
The novel Spergualin-related compounds represented
by the general formula ~I3 can form a salt with an acid.
Such acid can be any inorganic or organic acid as long as
it is nontoxic. There is no limitation for inorganic
acids; however, hydrochloric acid, sulfuric acid, nitric
acid, phosphoric acid, etc. are preferred. There is no
limitation for organic acids, either; however, the preferred
-- 5 --
l are acetic acid, propionic acid, succinic acid, fumaric
acid, maleic acid, malic acid, tartaric acid, glutaric
acid, citric acid, benzenesulfonic acid, toluenesulfonic
acid, methanesulfonic acid, ethanesulfonic acid, propane-
sulfonic acid, aspartic acid, glutamic acid, etc.
Examples of the compounds of the general formula
[I] according to the present invention ara shown in
Table l. In the present specification, abbreviations
have the following definitions.
TAD : triazadecane
GP : guanidinophenyl
GHep : guanidinoheptanoyl
For : 4-hydroxymathyl-3~hydroxyphenylglycyl
Amino acid residues
Ala : alanyl
Leu : leucyl
Phe : phenylalanyl
Asp : aspartyl
Asn : asparaginyl
Lys : lysyl
PhG : phenylglycyl
Pro : prolyl
Tyr : tyrosyl
Ser : seryl
~-Ala: ~-alanyl
AHPA : 3-amino-2-hydroxy-4-phenylbutyryl
Gly : glycyl
Glu : glutamyl
-- 6
1305~;~7
1 y-ABA: y-aminobutyryl
Protective groups
Z : benzyloxycarbonyl group
. Boc : t-butyloxycarbonyl group
pMZ : p-methoxybenzyloxycarbonyl group
Aoc : t-amyloxycarbonyl group
Amino acid residues containing protective groups
Asp(OBzl): ~-benzylaspartyl
Asp(OBut): ~-t-butylaspartyl
Ser(Bzl) : O-benzylseryl
Ser(But) : O-t-butylseryl
Z-Lys : ~-benzyloxycarbonyllysyl
Tyr(Bu ) : O-t-butyltyrosyl
5~
~o
~^ ~ 1~ ~
~i '~
~ 1~
-- 8
~L~Q5~Z7
.
o'l c~ ~ ~
. .. .. ~
O O _ V
$ X l ,
; o ~--æ ::~ v--æ
I ~ ~ V
--æ ~ o o
.. _ .- I
_ _
X ~ = = =
-
/~ o æ ~r ~ ~ r~
I V ~ . _ 9 _ _
~3~5
I ~C _
o'
~,~
_~ ,~ ~
o o .~ o
x _ m x o u
C~ ~: ~:: Z~
~ ~ ~1
-- 10 --
5~7
~o __~_ _ ~
o¢ U~ ~ ~ ~
_~ _ t~7 ~., .
~¢ ~ I 7 _~
0~ -' ~ f7
t~l~ C)-- ,'ri 5 1 -- l
~; X Cs~ ~ O ~ )
~ Z X C.) ~zX~ Z,~,
. a
lY P:: _ I C ~ ~:
[~ ~u
. .... __ .1
/la oz ..__ 7 - .._
-- 11 --
V5~7
U,~,c ,, _ _ , ,
o~ ~ ~ ~
_. o ~ ~ .
~, ~ o ~ ~
. :C U ~ _ U-Z
P~ ~:~ _ :~ o ~C
; a~ à'
...
~ ~ ~
~ .
. ¦u ~ . _
-- 12 --
1305~`7
__~ _
,
. __
X ~ . ~
_ _ . . ._ _ _
~"z . _
-- 13 --
5~7
/, ~
L
-- 14 --
~;~t?~ 7
I
~ I .,
~,
.
~o
Z '--U
U
.
o
~,
~; Z--U
X
X
'~ .
a~z ~
~_~ - 15 -
~3Q.~7
1 Of the compounds shown in Table 1, the preferable
are Compound Nos. 1, 2, 4 to 8, 13 and 14. L~lore
preferable are Compound Nos. 1, 2, 5, 13 and 14.
The compounds of the general formula [I] can
be synthesized by removing protecting groups from a
protecting group-containing compound represented by the
general formula [II]
H2N IR3 l4
6~C-NH-X-(CH2)3-CONHCHCONH-(CH2)4_N_(CH2~3_NH_R5 [II]
[wherein X is -(CH2)3_5- or _ ~ ; R3 is -H or -CH2-O-Y
(wherein Y is -H or a protecting group for -OH); R4 is -H,
a phenylglycyl group wherein the amino group is protected,
or a leucyl group wherein the amino group is protected;
and R5 is a residue obtained by removing, from an amino
acid or peptide having protected amino groups, the hydroxyl
group of the carboxyl group (the side chains of the
residue may be protected) and, when R4 is a phenylglycyl
group wherein the amino group is protected or a leucyl
group wherein the amino group protected, R5 is a phenyl-
glycyl or leucyl group wherein the amino group is protected.].
The removal of protecting groups can be
conducted by means such as reduction, acid decomposition,
hydrolysis or the like.
The reaction for removal of protecting groups
is ordinarily conducted in an inert solvent at temperatures
- 16 -
~;~05&~7
l from -60C to the boiling point of the solvent, preferably
from about -50C to about 100C. As the inert solvent,
there can be used water as well as hydrophilic organic
solvents such as lower alcohols (e.g. methanol, ethanol),
ketones (e.g. acetone, methyl ethyl ketone), amides
(e.g. dimethylformamide, dimethylacetamide), cyclic ethers
(e.g. tetrahydrofuran, dioxane), lower fatty acids (e.g.
acetic acid, trifluoroacetic acid~, liquid ammonia, liquid
hydrogen fluoride and the like.
After the completion of the reactlon for
removal of protecting group, the reaction mixture is
subjected to the following purification to isolate a novel
Spergualin-related compound of the general formula [I].
For example, when the removal of protecting group is
conducted by catalytic reduction using palladium black,
the reaction mixture is filtered to remove the catalyst;
the filtrate is concentrated under vacuum; and the residue
is purified by a known purification method using CM-
Sephadex ~ ~Na+) and Sephadex ~ LH-20 ~T. Takeuchi et al.,
J. Antibiotics, 34, 1619 (1981)] to obtain a desired
compound. When the removal of protecting group is conducted
using trifluoroacetic acid, the reaction mixture is
concentrated under vacuum and the residue is purified by
the same purification method as mentioned above to obtain
a desired compound.
In the above purification, the novel Spergualin-
related compound of the general formula [I] is o~tained
as a hydrochloride. This hydrochloride can be converted
- 17 -
t~
1 to other salt according to, for example, one of the
following procedures. The hydrochloride is dissolved in
water; the aqueous solution is passed through a strongly
basic ion exchange resin to collect a fraction containing
a desired compound; to the fraction is added a desired
acid, an aqueous solution containing the acid, or a solu-
tion of the acid dissolved in a hydrophilic organic acid
(e.g. methanol, ethanol, acetone, tetrahydrofuran, dioxane)
to effect neutralization; and the neutralization mixture
is subjected to dryness under vacuum orr when containing
an organic solvent, is subjected to vacuum distillation
to remove the solvent and then to lyophilization. Alter-
natively, the hydrochloride is mixed with silver hydroxide
or an aqueous silver oxide solution to neutralize the
hydrogen chloride of the hydrochloride; the reaction
mixture is filtered to remove the insoluble silver chloride;
the filtrate is mixed with a desired acid to form d salt;
and the reaction mixture is subjected to lyophilization.
The salt other than hydrochloride, obtained
as above may take a form of hydrate, depending upon the
treating conditions.
The protected Spergualin-related compounds of
the general formula [II] used as a starting material in
the present invention can be synthesized as follows.
- 18 -
1 ~a) Synthesis of compounds of the general formula [II]
wherein R4 is a hydrogen atom and R5 is a residue
obtained by removing, from an amino acid having
protected amino groups, the hydroxyl group of the
carboxyl group (the side chains of the residue may
be protected).
A Spergualin-related compound represented by
the general formula [III~.
H2N~
~C--NX-X-(CH2)3-co-NHc~coNH-(cH2)4--NH(CH2)3--NH2 [III]
(wherein X and R each have the same definition as given
previously) which is obtained according to a process
described in Japanese Patent Application Kokai (Laid-Open)
Nos. 42356/1984 and 185758/1985 is condensed with an
N-protected-- or ~-amino acid represented by the general
formula [IV]
R5-OH [IV]
[wherein R5 is a residue obtained by removing, from an
amino acid having protected amino groups, the hydroxyl
group of the carboxyl group (the side chains of the
residue may be protected)] or a reactive derivative
thereof to obtain a titled compound.
-- 19 --
~3~
1 (b) Synthesis o~ compounds of the general formula [II
wherein R4 ls a hydrogen atom and R5 is a residue
obtained by removing, from a peptide having protected
amino groups, the hydroxyl group of the carbo~yl
group (the side chains of the residue may be protected).
A compound of the general formula [III] is
condensed with a first N-protected amino acid or a
reactive derivative thereof and then the N-protecting
group is removed; the resulting compound is condensed
with a second N-protected amino acid or a reactive
derivative thereof and then the N-pxotecting group is
removed; whereby, a compound of the general formula [II]
wherein R5 is a residue obtained by removing, from an
N-protected dipeptide, the hydroxyl group of the carboxyl
group can be obtained.
When the compound of the general formula [II]
is further condensed with a third N-protected amino acid
and the N-protecting group is removed, a compound of
the general formula ~II] wherein R5 is a residue obtained
by removiny, from an N-protected tripeptide, the hydroxyl
group of the carboxyl group, can be obtained.
(c) Synthesis of compounds of the general formula [II]
wherein both R4 and R5 are an N-protected phenylglycyl
group or N-protected leucyl group.
A compound represented by the general formula
[V]
- 20 -
'7
NH2-(cH2~4-l-(cH2)3 NH R5 [V]
R4
1 (wherein both R4 and R5 are an N-protected phenylglycyl
group or an N-protected leucyl group) is condensed with
a protected amino acid represented by the general formula
[VI]
IR3
P2-NHCHCOOH [VI]
(wherein P2 is a protecting group and R3 has the same
definition as given previously) or a reactive derivatlve
thereof to obtain a compound represented by the general
formula [VII]
IR3
2 ( 2)4 1 (CH2)3 NH R5 [VII]
R4
(wherein P2, R3, R4 and R5 each have the same definition
as given previously). Then, the protecting group P2 is
removed selectively, and the resulting compound is condensed
with an ~-guanidino acid derivative represented by the
- general formula [VIII]
H2N~
~c-NH-x-(cH2)3-cooH [VIII]
(wherein X has the same definition as given previously) or
- 21 -
1305~7
1 a reactive derivative thereof to obtain a titled compound.
For information, the compound of the general
formula [V] can be produced according to the following
ordinary process.
For instance, 1 mole of an N-protected spermidine
represented by the general formula [IX]
Pl NH (CH2)4-NH-(cH2)3NH2 [IX]
(wherein Pl has the same definition as given previously)
is condensed with 1 mole of a reactive derivative
obtained from the reaction of an N-protected amino acid
of the general formula [IV]
R5 - OH [IV]
(wherein R5 has the same definition as qiven previously)
and 1,3-thiazolidine-2-thione; the condensation product
is then reacted with an N-protected amino acid rep--esented
by the general formula [X]
R4 - OH [X]
lS [wherein R4 is a residue obtained by removing -OH from an
amino acid having protected amino groups (the side chains
of the amino acid may be protected)]; subsequently, the
protecting group Pl is removed selectively to obtain a
compound of the general formula [V].
When R4 and R5 are same, the compound of the
- 22 -
i3~)5&X7
1 general formula [V] can also be produced by reacting 1
mole of an N-protected spermidine of the general formula
[IX] with at least 2 moles of a protected amino acid of
the general formula [IV] or [X] or a reactive derivative
thereof and then selectively removing the protecting
group Pl from the reaction product.
The N-protected spermidine of the general formula
[IX] can be produced by reacting a compound represented
by the general formula [XI]
1 NH(CH2)4NH2 [XI]
(wherein Pl has the same definition as given previously)
with acrylonitrile and then reducing the nitrile group,
The condensation in the above (a), (b) and (c)
can be conducted in accordance with an ordinary method
used for peptide linkage formation. The method includes
a carbodiimide method using dicyclohexylcarbodiimide,
l-ethyl-3-(N,N-dimethylaminopropyl)carbodiimide, etc.;
an azide method using hydrazide, etc.; a mixed acid
anhydride method using ethyl chlorocarbonate, isobutyl
chlorocarbonate, etc.; an active ester method using a
cyanomethyl ester, a vinyl ester, a substituted or
unsubstituted phenyl ester, a thiophenyl ester, a hydroxy-
succinimide ester, etc.; an o-acylhydroxylamine derivative
method using acetoxime, cyclohexanoxime, etc.; an N-acyl
compound method using carbonyldiimidazole, etc.; and a
carboxylic acid activation method using 1,3-thiazolidine~
- 23 -
32~
1 2-thione.
The solvent used in the condensation can be
those used in ordinary reaction of peptide linkage
formation. As the solvent, there can be mentioned, for
example, ethers such as diethyl ether, tetrahydrofuran
and the like; esters such as ethyl acetate and the
like; ketones such as acetone, methyl ethyl ketone and
the like; halogenated hydrocarbons such as methylene
chloride, chloroform and the like; amides such as
dimethylformamide, dimethylacetamide and the like; and
nitriles such as acetonitrile and the like. These solvents
can be used singly or, when they are miscible with water,
as a mixed solvent with water.
The protectlng groups for amino groups, used
lS in the present invention includes benzyloxycarbonyl
group, substituted benzyloxycarbonyl group (e.g. p-
methoxybenzyloxycarbonyl group), t-butyloxycarbonyl group,
t-amyloxycarbonyl group, formyl group, trityl group, o-
nitrophenylsulfenyl group, etc.
The protecting group for side chains of amino
acid can be as follows. The protecting group for carboxyl
group includes lower alkyl group, t-butyl group, benzyl
group and substituted benzyl group; the protecting group
for hydroxyl group includes t-butyl group and benzyl
group; the protecting group for mercapto group includes
benzyl group and p-methoxybenzyl group; the protecting
group for imidazole group includes benzyloxycarbonyl
group, benzyl group and tosyl group; and the protecting
- 24 -
l;~Q58;~
1 group for guanidino group includes nitro group, tosyl
group and t-butyloxycarbonyl group. However, the
protecting group for the side chains of amino acid is
not restricted to those mentioned above.
S Typical examples of the compound of the general
formula [II] used as a starting material in the present
invention are shown in Table 2. The stereochemical configu-
ration of amino acid residue is L, D or LD
Amino acid residues
Ala : alanyl
Leu : leucyl
Phe : phenylalanyl
Asp : aspartyl
Asn : asparaginyl
Lys : lysyl
PhG : phenylglycyl
Pro : prolyl
Tyr : tyrosyl
Ser : seryl
~-Ala: ~-alanyl
AHPA : 3-amino-2-hydroxy-4-phenylbutyryl
Gly : glycyl
Glu : glutamyl
y-ABA: y-aminobutyryl
Protective groups
Z : benzyloxycarbonyl group
~Q~5~7
1 Boc : t-butyloxycarbonyl group
pMZ : p methoxybenzyloxycarbonyl group
Aoc : t-amyloxycarbonyl group
Amino acid residues containing protective groups
Asp(OBzl): ~-benzylaspartyl
Asp(OBu ): ~-t-butylaspartyl
Ser(Bzl) : O-benzylseryl
Ser(Bu ) : o-t-butylseryl
Z-Lys : -benzyloxycarbonyllysyl
Tyr(Bu ) : O-t-butyltyrosyl
Table 2
Typical examples of the compound represented
by the general formula [II]
13 IR4
HN ~ 2)3 CONHCH-CONH-(CH2)4-N-(CH ) NH
_
X R3 R4 R5
-CH20H . Z-Leu-
,. Boc-Leu-
. " .- ll pMz-Leu-
.. _ _
., .. ,~ Z-PhG-
. .. .. Boc-PhG-
_ _ pMz-PhG-
~ 26 -
1~051~7
_ _ _ _ R4 R5
._._ ~ .. ..
-CH20H H Z-Phe-
., Boc-Phe-
.. .. pMz-Phe-
_. Z-Tyr-
.. ll Aoc-Tyr(But)-
ll ll 1. pMz-Tyr(But)-
.. .. 1. Z-Asp(OBzl)-
.. 1. ll Boc-Asp(OBut)-
pMz-Asp(OBut)-
. - Z-Ala-
. 1. 1. Boc-Ala-
.. ll ll pMz-Ala-
__ . _
Z-Ser(Bz.l)-
.. ll .. Boc-Ser(But)-
.. . ll pMz-Ser(But)-
.. .. . - Z-Pro-
.. ll .. Boc-Pro-
.. .. .. pMz-Pro-
__ ._
.. . ll Z-Asn-
.. ll ll Boc-Asn-
_ _ pMZ-Asn-
- 27 -
~L3~ 7
__ _ R3 R4 R5
z_
-CH20H H Z-Lys-
,- ,. Boc-Lys-
' _ " ,. pMz-Lys-
,. ll ll Z-Leu-Leu-
,. ll Boc-Leu-Leu-
.. ll pMz-Leu-Leu-
. _._ ._._ _ _ .. _
.. ll Z-PhG-PhG-
Boc-PhG-PhG-
_ _ _ pMz-PhG-PhG-
" Z-B-Ala-
" . ll Boc-~-Ala-
,. ll ll pMz-~-Ala-
_.
,- " . ll Z-AHPA-
- . Boc- AHPA-
.i pMz-AHPA-
H . " Z-Leu-
Boc-Leu-
pMz-Leu-
._ . .. _
Z-PhG-
,~ ll ll Boc-PhG-
_ _ _ _ _ _ pMz-PhG-
- 28 -
~305l~27
. .
R3 R4 R5
H H Z-Leu-Leu-
" ~. ll Boc-Leu-Leu-
" ll .. pMZ-Leu-Leu-
. __. _ . _~
ll H ll Z-Phe-
.. .. .. Boc-Phe-
.. .. ., pMz-Phe-
. . .
t CH2t3 -CH20H ,l Z-Leu-
ll ll ll Boc-Leu-
., ,~ ll pMz-Leu-
.. .. - Z-PhG-
.. .. .. Boc-PhG-
.. .. .. pMz-PhG-
.. .. _
Z-Leu-Leu-
Boc-Leu-Leu-
,. ll " pMz-Leu-Leu-
,. ll ,. Z-GABA
Z-Gly
.. Boc-Asp (OB2Q)
_ ___
H ll Z-Leu-Leu-
Boc-Leu-Leu-
.. .. pMz-Leu-Leu-
Z-Leu-
. ll Boc-Leu-
pMz-Leu-
- 29 -
~.305&~
R3 4 _ 5
t CH 2 ~ - 3 H H Z-PhG-
,~ .. .. Boc-PhG-
.. .. .. pMz-PhG-
_. _ .. _
-CH20Bzl Z-PhG- Z-PhG-
~-CH2-OBu Boc-PhG- . Boc-PhG-
.. ., pMz-PhG- pMz-PhG-
_ _ .
..-CH20Bzl Z-Leu- Z-Leu-
"-CH20BU Boc-Leu- Boc-Leu-
.. ,. pMz-Leu- pMz-Leu-
. ... _
...-C~20Bzl Z-Leu-Leu- Z-Leu-Leu-
..-CH2Bu Boc-Leu-Leu- Boc-Leu-Leu-
.. . pM -Leu-Leu- pMz-Leu-Leu-
.. _ ..
.. H Z-PhG- Z-PhG-
.. .. Boc-PhG- Boc-PhG-
.- .. pMz-PhG- pMz-PhG-
. _
t CH2~-3 .. Z-PhG- Z-PhG-
.. .. Boc-PhG- Boc-PhG-
. .. pMz-PhG- pMz-PhG-
.___ .... _
" -CH20Bzl Z-PhG- Z-PhG-
.. -CH20But Boc-PhG- Boc-PhG-
__ _ pMz-PhG- pMz-PhG-
- 30 ~
~.~0~3Z7
1 In using the present compounds as a medicine,
they are made into a desired preparation according to an
ordinary method, using, if necessary, an excipient and
can be administered orally or parenterally.
When an injection is prepared, it is advisable
that the injection ordinarily contain 0.1 to 30~ by
weight, preferably 1 to 10~ by weight of an active
ingredient. In oral administration, a tablet, a capsule,
a powder, a granule, a liquid, a dry syrup, etc. are used.
The capsule, the granule and the powder generally contains
5 to 100~ by weight, preferably 25 to 100% by-weight of
an active ingreident.
The amount of administration is determined by
the age and weight of patient, the condition of disease,
the purpose of cure, etc. but generally is 1 to 100
mg/kg/day in parenteral administration and 5 to 500
mg/kg/day in oral administration.
Next, the toxicity and physiological activity
of the present compound will be shown by experiments.
1. Methods of experiments
(a) Toxicity
A compound of the present invention was dissolved
in a physiological saline solution in various concentra-
tions. Each of the resulting solutions was intraperitoneally
administered to one of CDFl-SLC female mice (one group:
two to three mice) in an amount of 0.1 ml per 10 g of body
weight. The highest concentration of present compound in
- 31 -
~305~7
1 solution was ~00 mg/10 ml/kg, and starting from this
solution of highest concentration, solutions of lower
concentrations were prepared each time at a common ratio
of 2. The administration amount of present compound with
the lowest concentration solution with which at least
one mouse died was taken as an administration amount of
acute toxicity.
(b) Inhibitory activity against antibody formation
Test groups and a control group of CDFl-SLC
female mice (each group: five mice) were intravenously
sensitized with 1 x 108/0.2 ml of sheep red blood cells
(SRBC). A compound of the present invention was dis-
solved in a physiological saline solution in various
concentrations. Each of the resulting solutions was
administered to one of the test groups once a day for
three consecutive days from the next day of the sensitiza-
tion, in an amount of 0.1 ml/10 g (body weight)/day.
The physiological saline solution was administered to the
control group.
In the fourth day from the sensitization, all
the mice were killed and the number of anti-SRBC plaque-
forming cells (PFC) in the spleen cells of each mouse
was measured. From this number, the PFC number per 10
of spleen cells was calculated. The effect of the present
compound was expressed as an inhibition percentage (%) of
PFC number of test group as compared with control group.
- 32 -
58;~7
Inhibition percentage (~)
= [1 - (PFC number of test group)/
(PFC number of control group)3 x 100
1 (3) Life prolongation effect of present compound to
mouse leukemia cells L1210 and its toxicity
1 x 105/0.2 ml/mouse of mouse leukemia cells
L1210 were intraperitoneally transplanted to test groups
and a control group of CDFl-SLC female mice (each group:
four mice~. A compound of the present invention was
dissolved in a physiological saline solution in various
concentrations and each of the resulting solutions was
administered to one of the test groups once a day for
nine consecutive days from the next day of the trans-
plantation, in an amount of 0.1 ml/10 g (body weight)/day.
The physiological saline solution was administered to the
control group.
All the mice of the test groups and the control
group were observed from the next day of the L1210
transplantation to examine their days of survival, and
the average days of survival (T~ of each test group and
the average days of survival (C) of the control group were
calculated. The life prolongation percentage of the
present compound was expressed as (T/C) x 100.
It is believed that a (T/C) x 100 of 125 or more
is effective.
- 33 -
05827
1 2. Results of experiments
The administration amounts of present compounds
causing acute death when administered to mice are shown
in Table 3. The inhibitory actions for antibody formation,
S of typical compounds of the present invention are shown
in Table 4. The antitumor activities of typical compounds
of the present invention are shown in Table S. In Tables
4 and 5, a control compound is included. This compound
is such that in the general formula [I], X is ~ ,
R is -CH2OH and both of Rl and R2 are -H.
Table 3
Administration amounts of present compounds
causing acute death to mice
Compound No. (mg/kg)
1 100
3 100
4 100
100
6 100
7 100
9 > 100
100
12 100
13 100
14 200
-
- 34 -
1~5~3~7
Table 4
Inhibitory activlties against antibody formation,
o~ present compounds
~ . _ . ~
Inhibitory percentage
. . _
Compound No. Administration Administration
amount amount
1.56 mg/kg 6.25 mg/kg
_ _ _
4 68 95
13 53 88
14 -50* -75*
Contrcl
_ ~9 ~
* - ~minus) shows an increase of antibody
formation.
- 35 -
1~0~
Table S
Life prolongation effect of present compounds
to mouse leukemia L1210
. . ~
Administration
Compound No. amount T/C x 100 (%)
mg/lcg/day
_ . _ _
50.00 327
25.00 294
12.50 278
1 6.25 315
3.13 269
1.56 296
0.78 195
0.39 146
... _ __ .
50.00 53
25.00 281
12.50 272
6.25 300
2 3.13 331
1.56 244
0.78 152
0.39 136
_
50.00 197
25.00 247
12.50 243
4 6.25 237
3.13 228
1.56 195
- 36 -
"7
0.78 149
_ _ 0.39 125
S0 ~ 00 253
25.00 284
12.50 290
6.25 290
3.13 265
1.56 146
0.78 133
0.39 123
...._
50.00 162
25.00 270
`12.50 250
6 6.25 250
3.13 267
1.56 201
0.78 158
0.39 125
_
50.00 284
25.00 284
12.50 265
6.25 216
7 3.13 152
1.56 99
0.78 86
0.39 96
.. . _
- 37 -
l~O~ 7
~5.00 362
12.50 290
3 l35 3272
8 1.56 164
0.78 130
0.39 102
0.20 109
. .. _ _
100.00 103
50.00 250
25.00 241
9 6 25 276
3.13 . 155
1.56 125
0.78 118
.__
50.00 303
25.00 274
12.50 257
13 6.25 247
3.13 283
1.56 201
0.78 149
_ 0.39 132
- 38 -
Control 25.00 191
12.50 261
X= ~ 6.25 254
3.13 243
R=-CH2OH 1.56 211
0.78 158
Rl-R2=H 0.39 129
0.20 109
- . : ..
1 As is cle~r from the above experiments, the
administration amounts of present compounds causing acute
death are larger and accordingly they have higher safety.
Moreover, the present compounds have excellent physio-
logical activity. Therefore, the present compounds are
expected as medicines such as an immunosuppressive agent
and an antitumor agent. Of the present compounds, such
compounds as R2 of the general formula [I] is a neutral
amino acid or a peptide consisting of neutral amino
acids are preferred because of their excellent activities
and low toxicity.
Next, the present invention will be explained
concretely by way of Examples. In the Examples, the Rf
value of TLC (thin lzyer chromatography) was obtained
by using a silica gel 60 F254 plate (thickness: 0.25 mm)
manufactured by Merck Co., developing a sample containing
- 39 -
~ .~
~30S~7
1 a present compound ~or a distance of about 8 cm with adevelopin~ solution described later, measuring a
distance from the starting point to a center of the spot
of an intended compound (the present compound) and
dividing the distance by a distance from the starting
point to the front end of the developing solvent.
Detections were carried out with ultraviolet absorption,
or visualized with ninhydrin and a Sakaguchi reagent.
Example 1 10-{N-[4-(4-GP)butanoyl]-L-Ser}-l-L-PhG-
1,5,10-TAD trihydrochloride (compound No. 1)
0.76 g (0.98 mmol) of a white crystal of 10-
{N-[4-(4-GP)butanoyl]-L-Ser3-1-(Z-L-PhG)-1,5,10-TAD
dihydrochloride was dissolved in 30 ml of methanol.
Thereto was added 0.15 g of palladium black. The mixture
was subjected to catalytic reduction for 5 hr at room
temperature at normal pressure.
After the reaction, the reaction mixture was
filtered to remove the catalyst. The filtrate was
concentrated under vacuum to obtain 0.7 g of an oily
substance. (The yield was quantitative~)
This oily substance was dissolved in 6 ml of
distilled water and subjected to chromatography on a
column packed with 105 ml of CM-Sephadex ~ C-25 (Na ).
Then, elution was conducted in accordance with a gradient
elution method from 500 ml of distilled water to 500 ml
of an aqueous l.OM sodium chloride solution to collect
fractions containing a desired compound. These fractions
- 40 -
l;~OS~2~7
1 were concentrated to dryness under vacuum. Methanol was
added to the residue and the resulting solution was
filtered to remove sodium chloride. The resulting oily
substance was purified as follows to obtain a desired
compound.
In order to remove a small amount of remaining
sodium chloride/ the oily substance was dissolved in 4 ml
of methanol and subjected to chromatography on a column
packed with 70 ml of Sephadex ~ LX-20. Elution was
conducted with methanol to collect fractions containing
a desired compound. They were concentrated under vacuum.
In order to further remove a slight amount of impurities,
the resulting oily substance was dissolved in 4 ml of
distilled water and subjected to column chromatography
on a column packed with 70 ml of HP-20 ~ (Mitsubishi
Chemical Industries, Ltd.). Elution was conducted with
distilled water to collect fractions containing a desired
compound. The fractions were concentrated under vacuum.
The resulting oily substance was dissolved in 5 ml of
distilled water and the solution was filtered to remove
insolubles. The filtrate was lyophilized to obtain 0.37
g (yield: 55.63~ of a desired compound.
NMR (D2O, external TMS)
~ = 1.6-4.0 (m, 21H), 4.1-4.5 (d, 2H, J=5Hz),
4.6-4.9 (t, H, J=5Hz), 5.63 (s, H),
7.5-8.1 (m, 4H), 8.05 (s, 5H)
IR (KBr)
v ~cm~l) = 3325, 2950, 1650, 1510, 1250
~ 41 -
5&~
TLC (chloroform:methanol:aqueous 17% ammonia
solution - 6:~:1 v~v)
Rf = 0.15
[ ]20 +15.3 (C=1.03, H2O)
In Examples 2 to 24, other compounds of the
general formula [I] were produced from other compounds
of the genexal formula [II] in manners similar to that of
Example 1,
In Examples 2 to 24, if the amino protecting
group of l-amino acid of the compound represented by the
general formula [II] was not benzyloxycarbonyl group
but t-butyloxycarbonyl group, p-methoxybenzyloxycarbonyl
group or t-amyloxycarbonyl group, and the protecting
group for the carboxyl group or hydroxyl group was t-butyl
group, the treatment for removal of protecting group was
conducted not by catalytic reduction but by (a) tri-
fluoroacetic acid or, when the compound of the general
formula [II] contained both benzyloxycarbonyl group and
t-butyl group, (b) catalytic reduction followed by
trifluoroacetic acid; and the subsequent treatments were
conducted in the same manner as in Example 1 to obtain
respective desired compounds.
The results of Examples 2 to 24 are summarized
in Table 6.
- 42 -
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1;~05~27
1 Reference Exampl~ 1
Synthesis of 10-{N-[4-(4-GP)butanoyl]-L-Ser}-
1-(Boc-L-Leu)-1,5,10-TAD dihydrochloride
~1) 3-(N-Boc-L-Leu)-1,3-thiazolidine-2-thione
10 g (43.23 mmol) of N-Boc-L-leucine was dis-
solved in 100 ml of methylene chloride. Thereto was
added 5.15 g (43.23 mmol) of thiazolidine-2-thione.
Further, 8.92 g (43.23 mmol) of dicyclohexylcarbodiimide
was added with ice-cooling. The mixture was allowed
to react for 6 hours with ice-cooling. The reaction
mixture was filtered to remove precipitated N,N'-
dicyclohexylurea. The filtrate was concentrated under
vacuum to obtain a light yellow crystal. This crystal
was suspended in 40 ml of methanol. The suspension was
filtered to collect 5.38 g (yield: 41.32%) of the desired
compound.
IR (KBr)
v (cm 1) = 3380, 2930, 1675, 1510, 1335, 1250,
1160, 1040, 845, 755
(2) 10-{N-[4-(4-GP)butanoyl]-L-Ser}-l-(Boc-L-Leu)-
1,5,10-TAD dihydrochloride
300 mg (0.55 mmol) of a white crystal of
10-{N-[4-(4-GP)butanoyl]-L-Ser}-1,5,10-TAD trihydrochloride
was dissolved in 5 ml of methanol. Thereto was add~d
61.2 mg (0.61 mmol) of triethylamine with ice-cooling.
The mixture was alLowed to react for 10 minutes with
ice-cooling. To the reaction mixture was added 201 mg
- 56 -
S~2~
1 (0.61 mmol) of the light yellow crystal of 3-(N-Boc-L-
Leu)-1,3-thiazolidine-2-thione obtained in the above
(1). The resulting mixture was allowed to react
for 5 hours at room temperature. The reaction mixture
was concentrated under vacuum. The oily residue was
suspended in 30 ml of acetone. The supernatant was
discarded by decantation, and the same procedure was
repeated twice. The residue remained was concentrated
under vacuum to obtain 390 mg (yield: 98.2%) of a desired
compound as a white crystal.
IR (KBr)
v (cm 1) = 3280, 2950, 1640, 1510, 1365, 1250,
1165, 1045
TLC (chloroform:methanol:aqueous 17% ammonia
solution = 6:4:1 v/v)
Rf = 0.39
In Reference Examples 2 to 4, compounds of the
general formula [II] of the Examples except Example 14
were produced from various protected amino acids in the
same manner as in Reference Example 1.
Reference Example 2
Synthesis of 10-{N-[4-(4-GP)butanoyl]-L-Ser}-l-(Boc-
L-Leu-L-Leu)-1,5,10-TAD trihydrochloride
0.84 g (1.28 mmol) of the white crystal of
10-{N-[4-(4-GP~butanoyl]-L-Ser}-l-L-Leu-1,5,10-TAD
trihydrochloride obtained in Example 1 was subjected to
- 67 -
il ;~0~27
1 the same treatment as in Reference Example 1 (2) to
obtain the compound of the ~eneral formula [II~ of
E~ample 13.
Reference Example 3
Synthesis of 10-{N-[4-(4-GP)butanoyl]-O-benzyl-L-
Ser}-1,5-di-(N Boc-L-PhG)-1,5,10-TAD
(1) 10-Boc-1,5,10-TAD
18.9 g (100 mmol) of mono-N-Boc-1,4-butanediamine
[see Japanese Patent Application Kokai (Laid-Open) No.
10 192347/1982] was dissolved in 150 ml of chloroform. Thereto
was added 5.~7 g (105 mmol) of acrylonitrile with ice-
cooling, and the mixture was allowed to react for 3 days
at room temperature. The reaction mixture was concentrated
under vacuum to obtain 23.4 g of an oily substance.
The oily substance (23.4 g) was dissolved in
260 ml of an ammonia-saturated ethanol. Thereto was added
20 g of Raney nickel, and hydrogenation was effected for
5 hours at room temperature at 60 atm. After the reaction,
the reaction mixture was filtered to remove the catalyst.
The filtrate was concentrated under vacuum to obtain
23.7 g (yield: 96.7%) of a desired compound as an oily
substance.
NMR (D2O, external TMS)
~ = 1.6-2.5 (m, 6H), 1.9 (s, 9H), 2.7-3.3 (m, 6H),
3.4-3.8 (m, 2H)
- 68 -
S~27
1 (2) 10-Boc-1,5-di-(N-Z-L-PhG)-1,5,10-TAD
2.85 g (11.6 mmol) of the oily 10-Boc-1,5,10-
TAD obtained in the above (1) and 6.63 g (23.2 mmol) of
N-Z-L-phenylglycine were dissolved in 50 ml of dichloro-
methane. Thereto was added 5.3 g (27.84 mmol) of 1-
ethyl-3-(N,N'-dimethylaminopropyl)-carbodiimide hydro-
chloride with ice-cooling. The mixture was allowed to
react overnight at room temperature. The reaction
mixture was concentrated under vacuum to obtain an oily
substance. The oily substance was dissolved in 200 ml
of ethyl acetate. The solution was washed with an aqueous
5% sodium carbonate solution and an aqueous saturated
sodium chloride solution in this order. The organic layer
was dried over anhydrous sodium sulfate and the~ concen-
trated under vacuum to obtain 10.5 g of an oily substance.
The oily substance was subjected to columnchromatography using silica gel 60 manufactured by
Merck Co. Development by chloroform followed by elution
by a chloroform-methanol (20:1) mixture gave 4.9 g (yield:
54.44%) of an oily substance.
NMR (CDC13)
= 0.9-1.9 (m, 6H), 1.47 (s, 9H), 2.7-3.8 (m, 8H),
4.5-5.0 (b, H), 5.2-6.0 (m, 2H), 5.23 (s, 4H),
6.1-6.6 (b, 2H), 6.9-8.0 (b, H), 7.5 (s, lOH),
7.53 (s, lOH)
TLC (chloroform:methanol - 10:1 v/v)
Rf = 0.49
- 69 -
s~
1 (3) 1,5-Di~ Z-L-PhG)-1,5,10-TAD
4.9 g (6.28 mmol) of the 10-Boc-1,5-di-
(N-Z-L-PhG)-1,5,10-TAD obtained in the above (2) was
dissolved in 20 ml of trifluoroacetic acid with ice-
cooling. The solution was allowed to react for 2 hours.The reaction mixture was concentrated under vacuum to
obtain an oily substance. The oily substance was dis-
solved in 150 ml of ethyl acetate, and the solution was
washed with an aqueous 5~ sodium carbonate solution and
a saturated aqueous sodium chloride solution in this
order. The organic layer was dried over anhydrous sodium
sulfate and then concentrated under vacuum to obtain 4.4 g
(yield: quantitative) of a desired compound as an oily
substance.
5 IR (KBr)
v (cm 1) = 3290, 3030, 2920, 1700~ 1635, 1490,
1445, 1325, 1230, 1145, 1035
TLC (chloroform:methanol = 1:1 v/v)
Rf = 0.12
(4) 10-(N-Boc-O-benzyl-L-Ser)-1,5-di-(N-Z-L-PhG)-
1,5,10-TAD
3.2 g (4.7 mmol~ of the 1,5-di-(N-Z-L-PhG)-
1,5,10-TAD obtained in the above (3) was dissolved in 40 ml
of dichloromethane. Thereto was added 0.8 g (7.9 mmol)
of triethylamine with ice-cooling. Further, 2.39 g
(6.09 mmol) of N-Boc-O-benzyl-L-serine N-hydroxysuccinimide
ester was added. The mixture was allowed to react overnight
- 70 -
~05~2~7
1 at room temperature. The reaction mixture was concentrated
under vacuum and the residue was dissolved in 200 ml of
ethyl acetate. The solution was washed with 5% phosphoric
acid, an aqueous 5% sodium carbonate solution and a
saturated aqueous sodium chloride solution in this order.
The organic layer was dried over anhydrous sodium sulfate
and then filtered to remove the drying agent. The filtrate
was concentrated under vacuum to obtain 4.2 g (yield:
93.3~) of a desired compound as a light yellow oily
substance.
IR (KBr)
v (cm 1) = 3305, 2930, 1705, 1650, 1495, 1450,
1300, 1235, 1160, 1040
TLC (chloroform:methanol = 20:1 v/v)
Rf = 0.43
(5) 10-(O-Benzyl-L-Ser)-1,5-di-(N-Z-L-PhG)-1,5,10-TAD
4.2 g (4.38 mmol) of the 10-(N-Boc-O-benzyl-
L-Ser)-1,5-di-(N-Z-L-PhG)-1,5,10-TAD obtained in the above
(4) was dissolved in 20 ml of trifluoroacetic acid with
ice-cooling. The solution was allowed to react for 2
hours. The reaction mixture was concentrated under
vacuum to obtain an oily substance. This substance was
dissolved in 150 ml of ethyl acetate and washed with an
aqueous 5% sodium carbonate solution and a saturated
aqueous sodium chloride solution in this order. The
organic layer was dried over anhydrous sodium sulfate,
and then filtered to remove the drying agent. The filtrate
32~
1 was concentrated under vacuum to obtain 3.7 g (yield:
~uantitative) of a desired compound as an oily substance.
IR (K~r)
v (cm 1) = 3305, 2930, 1710, 1640, 1520, 1495,
1450, 1325, 1235, 1075, 1040
TLC (chloroform:methanol = 10:1 v/v)
Rf = 0.13
(6) 10-{N-[4-(4-GP)butanoyl]-O-benzyl-L-Ser}-1,5-di-
(N-Z-L-PhG)-1,5,10-TAD hydrochloride
1.2 g (5.42 mmol) of a brown crystal of 4-(4-
GP)butyric acid was added to 7 ml of ice-cooled thionyl
chloride, in 4 to 5 portions. The mixture was allowed
to react for 15 minutes. The reaction mixture was
concentrated to dryness under vacuum.
3.8 g (4.38 mmol) of the 10-(O-benzyl-L-Ser)-
1,5-di-(N-Z-L-PhG)-l,S,10-TAD obtained in the above (5)
was dissolved in 30 ml of dimethylformamide. Thereto
was added 0.65 g (8.21 mmol) of pyridine with ice-
cooling. There was further added a solution obtained by
dissolving a hydrochloride of the above prepared 4-(4-GP)-
butyric chloride in 7 ml of dimethylformamideO The
mixture was allowed to react for 30 minutes with ice-
cooling. The reaction mixture was concentrated under
vacuum to obtain an oily residue. The residue was dis-
solved in a mixture consisting of 300 ml of ethyl acetateand 90 ml of ethanol and the mixture was washed with 5%
phosphoric acid, an aqueous 5% sodium carbonate solution
- 72 -
~3~5~
1 and a saturated aqueous sodlum chloride solution in this
order. Slnce a slight amount of an oily substance
precipitated during washing, ethanol was added to dis-
solve the preciptate. The organic layer was dried over
anhydrous sodium sulfate and then filtered to remove
the drying agent. The filtrate was concentrated under
vacuum to obtain 4.8 g (yield: quantitative) of a desired
compound as a light yellow oily substance.
IR (KBr)
v (cm 1) = 3300, 2930, 1645, 1515, 1450, 1235,
1045
TLC (chloroform:methanol:aqueous 17~ ammonia solution
= 6:1.5:0.25 v/v)
RF = 0.28
Reference Example 4
Synthesis of 10-{N-[4-(4-GP)butanoyl]-L-Ser}-l-
(Z-L-PhG)-1,5,10-TAD dihydrochloride
(1) 3-(N-Z-L-PhG)-1,3-thiazolidine-2-thione
5.7 g (20 mmol) of N-Z-L-phenylglycine was
dissolved in 50 ml of methylene chloride~ Thereto was
added 2.38 g (20 mmol) of 1,3-thiazolidine-2-thione.
Further, 4.13 g (20 mmol) of dicyclohexylcarbodiimide
was added with ice-cooling. The mixture was allowed to
react for 6 hours with ice-cooling. The reaction mixture
was filtered to remove N,N'-dicyclohexylurea. The
filtrate was concentrated under vacuum to obtain 12.0 g
of a light yellow oily substance. The substance was
- 73 -
05~3~7
1 chromatographed on a column packed with silica ge ~ 60
manufactured by Merck Co. Development was conducted
using a mixed solvent, n-hexane-chloroform-ethyl
acetate (6 : 3 : 1-2 v/v) mixture, whereby 4.0 g (yield:
51.28%) of a desired compound was obtained as a light
yellow oily substance.
IR (KBr)
v (cm 1) = 3390, 1690, 1585, 1500, 1455, 1335,
1275, 1225, 1170, 1055
TLC (n-hexane:chloroform:ethyl acetate = 6:3:2 v/v)
Rf = 0.28
(2) 10-{N-[4-(4-GP)butanoyl]-L-Ser}-l-~Z-L-PhG)-1,5,10-
TAD dihydrochloride
0.55 g (1 mmol) of a white crystal of 10-{N-[4-
(4-GP)butanoyl]-L-Ser}-1,5,10-TAD trihydrochloride was
dissolved in 6 ml of methanol. Thereto was added 0.106 g
(1.05 mmol) of triethylamine with ice-cooling.
The mixture was allowed to react for 10 minutes
with ice-cooling. To the reaction mixture was added
0.41 g (1.05 mmol) of the light yellow oily substance of
3-(N-Z-L-PhG)-1,3-thiazolidine-2-thione obtained in the
above (1). The mixture was allowed to react for 5 hours
at room temperature. The reaction mixture was concentrated
under vacuum. The oily residue was suspended in 30 ml of
acetone. The supernatant was discarded by decantation.
The same procedures was repeated twice. The residue was
dried under vacuum to obtain 0.83 g (yield: quantitative)
- 74 -
~31~:~5&~7
1 of a desired compound as a white crystal.
IR (KBr)
~, (cm 1) = 3270, 1620, 1510, 1235, 1040
TLC (chloroform:methanol:aqueous 17~ ammonia solution
= 6:4:1 v/v)
Rf = 0.51
Various other compounds of the general formula
[II]-can be produced from various protected amino acids
in the same manner as in Reference Example 1.
When the product obtained in Reference Example
1 (1) is a light yellow crystal, further purification
with column chromatography is not necessary. The
crystal is suspended in methanol and is recovered by
filtration, whereby a desired compound can be obtained
with high purity.
